Lipid A, the hydrophobic membrane anchor of lipopolysaccharide, is a glucosamine-based saccharolipid that constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and protects bacteria from the external damage of detergents and antibiotics. It is also the active component of lipopolysaccharide that causes life-threatening Gram-negative septic shock. Lipid A is synthesized by nine enzymes of the Raetz pathway in E. coli. The first six enzymes of lipid A biosynthesis are required for the viability of virtually all Gram-negative bacteria and are novel antibiotic targets. After lipid A i generated, it is flipped from the cytosolic surface of the inner membrane to the periplasmic surface, where it can be further transformed by lipid A modification enzymes that are specific to individual bacterial species. These modifications help bacteria evade the host immune response, adapt to environmental changes, and generate altered lipid A molecules that display diverse bioactivities. Although detailed structural analyses of several lipid A biosynthetic and modification enzymes have been carried out, providing rich information for structure-aided inhibitor development, others require further characterization. This proposal focuses on biochemical and structural studies of the essential lipid A biosynthetic enzymes that convert UDP-diacylglucosamine (UDP-DAGn) to 2,3- diacyl-GlcN-1-P (lipid X) and the lipid A modification enzyme LpxE that is important for bacterial virulence and production of monophosphorylated lipid A (MPLA), a widely used immunotherapeutic agent. These studies are expected to generate fundamental insights into the structure and mechanism of lipid A enzymes as well as the biogenesis and function of the bacterial outer membrane, and ultimately contribute to the development of novel therapeutics to improve human health.